This invention relates to a novel method for the treatment of vascular leak and systemic inflammatory response syndrome (SIRS), which conditions include, e.g., sepsis syndrome, septic shock, nonresponsive septic shock, multiple organ dysfunction syndrome, multiple organ failure syndrome and shock resulting from immunologically mediated organ injury, pancreatitis, hemorrhage, ischemia or multitrauma. The invention further relates to a method for identifying patients at risk of developing vascular leakage syndrome and SIRS on the basis of levels of matrix metalloproteinase expression, in particular, expression of collagenases.
The invention further relates to in vitro methods for determining specific compounds which comprise utility in the treatment of vascular leakage syndrome and SIRS, such as septic shock and other metalloproteinase related disorders on the basis of their ability to inhibit the activity of matrix metalloproteinases or to inhibit the expression of matrix metalloproteinases.
Further, the invention provides in vitro methods for screening specific bisdioxopiperazine compounds those of which inhibit matrix metalloproteinases, in particular, type IV collagenases. The invention further relates to methods of using bis(dioxopiperazine) compounds for the treatment of metalloproteinase, in particular collagenase related disorders, such as vascular leakage syndrome and SIRS, particularly septic shock.
It is believed that there are about 400,000 cases of sepsis each year, and 200,000 cases of septic shock, with about 100,000 of such cases resulting in death annually. However, despite the prevalence of these disease conditions, available methods for treatment of sepsis and septic shock are ineffective, at least in part because the precise cause of these disease conditions has been poorly understood.
Sepsis essentially comprises clinical evidence of bacterial infection and symptoms characterized by (i) temperatures in excess of about 101.degree. F., or less than 96.degree. F., (ii) heart rates greater than 90 beats per minute, and (iii) respiratory rates in excess of about 20 breaths per minute. Such symptoms are often followed by hypothermia, nausea, vomiting and diarrhea.
Sepsis syndrome comprises sepsis together with evidence of altered organ perfusion. Such evidence may include one or more of (i) acute changes in mental states, (ii) Pa.sub.o.sbsb.2 /Fl.sub.o.sbsb.2 &lt;280, (iii) increased lactate levels, and (iv) oliguria (&lt;0.5 ml/Kg for at least one hour).
Septic shock essentially comprises sepsis syndrome together with hypotension which is responsive to intravenous fluids or pharmacological intervention, i.e., systolic blood pressure&lt;90 mm Hg or decrease in mean arterial pressure greater than 40 mm Hg in a hypertensive patient.
Nonresponsive septic shock differs from septic shock in that the hypotension lasts for greater than 1 hour, and is not responsive to intravenously administered fluids or pharmacological intervention.
Septic shock symptoms include tachycardia, hypotension, disseminated intravascular coagulation, and the leakage of plasma proteins into the tissues (vascular leakage syndrome) that may result in peripheral cyanosis, oliguria and edema. Death typically results from renal, respiratory or cardiac failure or a combination thereof.
As yet there is no good way to predict which patients exhibiting infection will develop inflammatory response syndrome (including sepsis) or multiple organ dysfunction syndrome. While some patients with severe infections may not develop sepsis, others, with relatively mild infections and/or symptoms of infection may develop a massive systemic response, i.e., inflammatory response syndrome (including sepsis) or multiple organ dysfunction syndrome.
However, sepsis is the major risk factor in the development of multiple organ failure syndrome. Sequential pulmonary, gastrointestinal, and renal failure may be recognized as early as 12 hours after the beginning of sepsis and septic shock or as late as 7 to 10 days. Because the risk of multiple organ failure syndrome increases with the severity and the duration of septic shock, (although it is not necessarily predictive) it is critically important to restore perfusion as rapidly as possible to avoid late morbidity and reduce the risk of mortality.
The exact pathophysiology of multiple organ failure syndrome is not known. It is hypothesized that injury of the microvascular system, especially the microvascular endothelium, is a factor common to ischemia-reperfusion injury and multiple organ failure syndrome. The microvascular endothelium is thought to be essential in the onset of this syndrome.
Several components of the immunoinflammatory defense system are implicated in the development of microvascular injury: neutrophil (PMN) activation, endothelial activation, complement activation (C5a), platelet-activating factor, arachidonic acid metabolites (LTB4, protacyclin, thromboxane), macrophage activation, the coagulation cascades, and cytokines (e.g., tumor necrosis factor, IL-1, interferon).
Currently available treatments for sepsis and septic shock involve the support of respiration, blood volume replacement, removal of infectious agents by surgery and/or antibiotics, use of vasoactive drugs drugs which increase renal or cardiac functions and glucocorticosteroids.
Although the cause of septic shock is not totally understood, recently various theories have been advanced as to the putative pathway for the induction and development of septic shock. It has been hypothesized that bacterial infection (bacteremia) results in the production of substances, in particular lipopolysaccharides (endotoxins) which induce the formation of cytokines, e.g., IL-1, TNF, PGES and other inflammatory mediators, which in turn result in the production of substances including, e.g., collagenases, nitric oxide and coagulation factors, which increase prior to the onset of vascular leakage syndrome and septic shock.
Currently known and proposed methods of treating septic shock have essentially attempted to kill the bacteria causing the infection, to reduce serum endotoxin levels to inhibit IL-1, TNF, PGES and other inflammatory mediators, or to inhibit nitric oxide or coagulation factors. However, none of the currently available or proposed treatments treats vascular leakage syndrome. This is despite the fact that vascular leakage syndrome, essentially the leakage of plasma proteins from blood vessels into the surrounding tissues, is related to the occurrence of the following symptoms: peripheral cyanosis, oliguria, edema and end-organ failure which may occur during the advanced stages of sepsis and may result in the death of the patient. Thus, a method for treatment of vascular leakage syndrome would be highly beneficial.
As discussed supra, it has been observed that matrix metalloproteinase levels, in particular collagenases such as type IV collagenases increase during sepsis and septic shock. Type IV collagenase enzymes are involved in the breakdown of type IV collagen, a major component of basement membrane, which accounts for about 20-70% of their total mass and forms a supramolecular network which maintains the integrity and rigidity of the basement membrane. Thus, disruption of the collagen IV network is believed to be a critical step in basement membrane degradation. Unlike other basement membrane components, collagen IV is highly resistant to proteolytic degradation by ubiquitous serine proteases but is susceptible to the action to specific metalloproteinases, in particular, the type IV collagenases.
Type IV collagenases are members of a large family of enzymes which include interstitial collagenases, stromelysin, and PUMP-1, which degrade extracellular matrix and basement membrane components. These enzymes all contain a zinc ion which is essential for proteolytic activity, and are secreted in a latent form (zymogen or proenzyme) requiring activation for proteolytic activity, (believed to be affected by plasmin) and are inhibited by a class of naturally occurring inhibitors known as tissue inhibitors of metalloproteinases (TIMP's).
Both normal and tumor cells produce two different but closely related metalloproteinases (MMP's) with type IV collagenase activity, namely, a 72 KD and a 92 KD enzyme respectively known in the literature as MMP-2 and MMP-9 which may be activated by p-AMPA in vitro and activated by plasmin in vivo. It is also known that cells produce natural inhibitors of these enzymes, e.g., TIMP-1 and TIMP-2. The active and inactive forms and substrates for these enzymes are set forth in Table I:
TABLE 1 ______________________________________ NOMENCLATURE AND NATURAL SUBSTRATES OF TYPE IV COLLAGENASES ENZYME NAMES MOLECULAR MASS SUBSTRATES ______________________________________ MMP-2 proenzyme 72 KD secreted gelatin MMP-2 enzyme 62-64 KD active gelatin collagens IV, V, VII, X fibronectin, elastin MMP-9 proenzyme 92 KD secreted gelatin MMP-9 enzyme 84 KD active gelatin collagens IV, V ______________________________________
The complete amino acid structure has been elucidated for MMP-2 and MMP-9 as well as their inactive prepro and pro-forms, and may be found in U.S. Pat. No. 4,992,537 to Goldberg et al.
The complete amino acid sequences for the endogenous tissue inhibitors of metalloproteinase, i.e., TIMP-1 and TIMP-2 are also known in the art. (DeClerk et al, J. Biol. Chem., 264, 17445 (1989); Boone et al, Proc. Natl. Acad. Sci., USA 87, 2800 (1990); Docherty et al, Nature, 318, 65 (1985); and Carmichael et al, Proc. Natl. Acad. Sci., USA, 83, 2407 (1986)).
The expression of matrix metalloproteinases and collagenases in particular have been studied by numerous research groups. For example, mononuclear phagocytes are known to synthesize and secrete a 57 kilodalton interstitial collagenase (MMP-1), a 60 kilodalton stromelysin (MMP-3), a 72 kilodalton type IV collagenase (MMP-2) and a 92 kilodalton type IV collagenase (MMP-9). (Wegus et al., J. Clin. Invest, 86, 1496, (1990)).
Additionally, mononuclear phagocyles are known to produce IL-1 and TNF which induces metalloproteinase gene expression (Dayer et al., J. Clin. Invest., 77, 645 (1986); Dayer et al., J. Exp. Med., 162, 2163 (1985)). Moreover, endotoxin has been disclosed to stimulate the synthesis and secretion of MMP-1, MMP-2, MMP-3 and MMP-4 from mononuclear phagocytes in vitro. (Wegus et al., J. Clin. Invest, 86, 1496 (1990)).
It has further been reported that TNF stimulates collagenase and PGE2 expression by human synovial cells (Dayer et al., J. Exp. Med., 162, 2163 (1985)), and that it stimulates the biosynthesis of MMP-9 in cultural human chorionic cells (So et al, Biol. Reprod., 46, 772 (1992)) and MMP-9 in osteosarcoma and fibrosarcoma cell lines (Okada et al, Biochem. Biophys. Res. Commun., 171, 610 (1990)). TNF has further been reported to inhibit collagen gene transcription and collagen synthesis in cultured human fibroblasts (Solis-Herruzo et al., J. Biol. Chem., 263, 841 (1988)).
However, to the best knowledge of the present inventors, there had been no recognition that the level of metalloproteinase expression, and in particular type IV collagenase expression, may be used as a means to predict the onset of vascular leakage syndrome or SIRS, e.g., septic shock. Moreover, there had been no recognition that the inhibition of these enzymes could be used to treat or prevent vascular leakage syndrome or SIRS such as septic shock.
As discussed supra, the invention further relates to the identification of particular bis(dioxopiperazine) compounds which inhibit matrix metalloproteinases, in particular collagenases, and the use thereof to treat collagenase mediated conditions or disorders.
Bis(dioxopiperazine)s and in particular 2,6-dioxopiperazines, are currently used in the treatment of various disease conditions, including, e.g., the treatment of cancer, inhibition of cardiac toxicity attributable to anti-cancer drugs such as doxorubicin, psoriasis, as radiation sensitizers during radiotherapy, as well as being proposed for the treatment of arthritis.
2,6-Dioxopiperazines are imides of open chain di(carboxymethyl)amines. The significance of 2,6-dioxopiperazine heterocyclic chemistry and biology evolved as a consequence of exploitation of ethylenediaminetetraacetic acid (EDTA; 1) pharmacology; cyclized imide derivatives of EDTA and related tetraacids are bis(2,6-dioxopiperazine)s wherein the amino N(4) nitrogens of each ring are connected by a central alkene side chain. Medicinal chemical investigations led to the discovery of these important drugs which possess synergistic antitumor, antimetastastic, and cardioprotective properties. For an exhaustive review of the chemistry and biology of regioisomeric dioxopiperazines, See, Witiak and Wei, In: Progress In Drug Research, E. Tucker, Ed. Vol. 35, Birkhauser Verlag Base, Boston, (1991), pp 249-363.
The bis(2,6-dioxopiperazine)s, first synthesized in the late 1950's, were thought to have potential for use as textile leveling agents or as pharmaceuticals, but no clear indication for their use was documented. Later, workers at the Imperial Cancer Research Fund (ICRF) introduced rationales for their use as antineoplastic agents providing the impetus for a world wide interest in exploring bis(2,6-dioxopiperazine)s as anti-cancer drugs. (See e.g., Nair et al, J. Chem. Ed., (1988), 65, 534-538; Creighton, U.S. Pat. No. 3,941,790, Creighton et al., Biochem. J. (1969), 114, 58P.)
Whereas many antitumor drugs are chelators of metal ions, EDTA (1) or its methyl (or ethyl) esters fail to demonstrate any significant antitumor activity. Reaction of EDTA, however, with formamide generates a diimide known as ICRF-154 (3) having weak antitumor properties in mice. (See Creighton: Prog. Antimicrob. Anticancer Chemotherapy, (1970), 1, 167-169; Creighton et al, Nature, (1969), 222, 384-385.
The structure of EDTA and methyl or ethyl esters thereof follows:
______________________________________ ##STR1## ##STR2## ______________________________________ 1; X = OH 3; R.sub.1 = R.sub.2 = H(ICRF-154) 2; X = NH.sub.2 4; R.sub.1 = Me, R.sub.2 = H(ICRF-159) 5; (+)isomer of ICRF-159(ICRF-187) 6; (-) isomer of ICRF-159(ICRF-186; also known as ADR-888) 7; R.sub.1 = Et, R.sub.2 = H(ICRF-192) 8; R.sub.1 = R.sub.2 = Me(meso isomer ICRF-193) 9; R.sub.1 = R.sub.2 = Me(dl isomer ICRF-196) 10; R.sub.1 = Me, R.sub.2 = n-Pr 11; R.sub.1 = Me, R.sub.2 = i-Pr 12; R.sub.1 = Et, R.sub.2 = n-Pr 13; R.sub.1 = Me, R.sub.2 = CH.sub.2 OMe(dl-erthro) 14; R.sub.1 = Me, R.sub.2 = CH.sub.2 OME(dl-threo) ______________________________________
The related propylene analogue ICRF-159 (razoxane, 4) effects a 93% (5 doses at 30 mg/kg) inhibition of S180 tumors in mice and a 137% (13 doses at 30 mg/kg) increase in survival time in the L1210 leukemia model (Creighton et al, Nature (1969), 222, 384-385). The (+) and (-) enantiomers of razoxane are known as ICRF-187 (5) and ICRF-186 (6), respectively. ID.sub.50 inhibition values for mouse L cell colony formation are for ICRF-154 (3) 7.3 .mu.M, razoxane (4) 3.0 .mu.M, ICRF-192 (7) 720 .mu.M, meso isomer ICRF-193 (8) 0.09 .mu.M, and racemic ICRF-196 (9) 150 .mu.M. Although meso bis(imide) 8 is more potent than razoxane in this assay, its therapeutic index is lower than that of razoxane in vivo. (Creighton et al, Proc. of the 6th Int. Symp. Med., (1978), pp. 281-288, Creighton et al, Nature, (1969), pp. 222, 384-385). The calculated ratio of maximum tolerated dose to the dose required for 90% inhibition of S180 tumor growth for ICRF-193=6.7; for razoxane this ratio=9.8.
Inhibition of .sup.3 H!thymidine incorporation into monolayers of mouse-embryo fibroblasts reveals IC.sub.50 values for ICRF-154=2.0 .mu.g/ml, razoxane=0.5 .mu.g/ml, meso ICRF-193=0.035 .mu.g/ml, dl-ICRF-196 (inactive), and a compound having a trans cyclobutanediyl linkage (ICRF-197)--14 g/ml, or a 1,3-propanediyl linkage (ICRF-161) (inactive). (Creighton, Prog. Antimicrob. Anticancer Chemother., (1970), 1, pp. 167-164). Other dl-erythro (10-13) and dl-threo (14) analogues of increased lipophilicity also have considerably decreased activity. (Creighton, Ger. Offer., (1972) 2, pp. 163, 601; Creighton, Prog. Antimicrob. Anticancer Chemother., (1970), 1, pp. 167-169).
Only minor modifications of the bis(imide) system are allowed in order to preserve biological activity. Two intact 2,6-dioxopiperazine moieties are thought to be important for biological activity. Replacement of 2,6-dioxopiperazine rings with other heterocycles, or hydrolysis to produce diamide diacids affords inactive materials, but these hydrolysis products are now thought to have biological significance. Generally, linkages between the two heterocyclic rings having five or fewer carbons produce compounds which are more effective in vitro.
Conformational mobility of the linking group leads to arrangements of dioxopiperazine rings, one or more of which likely is important for various biological activities. Crystal structure analysis of racemic razoxane (4) reveals a cis face-to-face relationship of dioxopiperazine rings; the (+) isomer ICRF-187 has the extended trans conformation with a parallel arrangement of ring planes in the solid state. (Hempel et al, J. Am. Cancer Soc. (1982), 104, 3453-3456). These conformations are mimicked by insertion of the 1,2-cyclopropanediyl spacer in place of the 1,2-propanediyl group of razoxane or its optical isomers. (Witiak et al, J. Med. Chem. (1978), 21, 1194-1197). The dioxopiperazine rings in these molecules, designated compounds 15 and 16, are held trans in 15 or cis as in 16. Biological properties of trans-15 and cis-16 differ markedly. The cis isomer inhibits, and the trans isomer stimulates development of metastases in two different animal models. (Witiak et al, J. Med. Chem., (1978), 21, pp. 1194-1197; Zwilling et al, Br. J. Cancer, (1981), 44, pp. 578-583). Cytostatic activity seems to reside in the cis conformation (see e.g., Witiak et al, J. Med. Chem., (1978), 21, 1194-1197; Zwilling et al, Br. J. Cancer, (1981), 44, 578-583), a conformation likely important to biometal chelation mechanisms, but solubility differences between the cis- and trans-1, 2-cyclopropanediyl analogues may also account for differences in observed activities. (Zwilling et al, Br. J. Cancer, (1981), 44, 578-583). The structures for these compounds are set forth on the following page: ##STR3##
The compound cis-16 is prepared from cis-1,2-cyclopropanediyl acid (17) convertible to diamine 18 which undergoes tetra-N-aLkylation to produce intermediate 19. (Witiak et al, J. Med. Chem., (1978), 21, pp. 1194-1197). Trans-15 is similarly synthesized from the trans isomer of 17. (Witiak et al, J. Med. Chem., (1978), 20, 630-635. These compounds are set forth below: ##STR4##
Razoxane has cytoxic activity against V-79A fibroblasts in tissue culture, but trans-15 is inactive. The decrease in activity of trans-15 in this system, and the reported inactivities of 9 and the trans-1,2-cyclobutanediyl bisimide in S180, leukemia L1210 and .sup.3 H!-thymidine assays may be related to similarities in their preferred geometries. Cis, not trans geometries, are anticipated to be preferred for most biological activities of bis(dioxopiperazine)s.
Bis(imide)s 20 and 21 are conformationally constrained analogues of ICRF-154 (3) and differ from 3 by two hydrogen atoms in their molecular weight. These geometric isomers are also related to cis-16; removal of the cyclopropyl methylene (CH.sub.2 function of 16 with concomitant bond connection between two C(3) carbons of the dioxopiperazine rings provides isomers trans-20 or cis-21. These tricycles are tetraazaperhydrophenanthrenes wherein the dioxopiperazine rings maintain a cisoid relationship. Formal bond disconnection of one carbonyl group from the central piperazine ring of 20 (or 21) and rebonding on the opposite carbon of the central ring provides diastereomers trans-22 and cis-23 belonging to the tetrazaperhydroanthracene series. The dioxopiperazines rings of geometric isomers 22 and 23 have an "extended" rather than "cisoid" geometry. These compounds are depicted below: ##STR5##
Tricyclic bis(2,6-dioxopiperazine)s 20 and 21 are prepared from pyrazine-2,3-dicarboxiamide (24) convertible to cis-25 (R=Me or Et) via reduction and N-alkylation. (Witiak et al, J. Med. Chem., (1981), 24, pp. 1329-1332). Cyclization of 25 in NaOMe/MeOH produces the trans isomer 20, but in NaOEt/EtOH cis-21 is the exclusive geometric isomer; product geometry is independent of starting esters and likely reflects solubility differences in MeOH vs. EtOH. In MeOH epimerization takes place affording the thermodynamically most stable trans isomer. These structures are set forth on the following page: ##STR6##
Bis(imide) isomers 22 and 23 may be synthesized following similar reaction sequences using 2,5-dimethylpyrazine (26) as starting material. (Witiak et al, J. Med. Chem., (1985), 28, pp. 1228-1234). Oxidation of methyl groups, ring reduction, and N-alkylation yield respective diastereomers 27 and 28 which are convertible to targets 22 and 23. ##STR7##
Pretreatment of B16-F10 melanoma cells with trans-20 significantly reduces the metastasis to the lungs of mice at all dose levels (2, 20 and 100 .mu.M), whereas the isomer cis-21 is ineffective in this model. (Witiak et al, J. Med. Chem., (1981), 24, pp. 1321-1332.) Regioisomeric bis(imide)s 22 and 23 have no antimetastatic effects against implanted Lewis lung LL carcinoma in mice, and these data again emphasize the need for a "cisoid" relationship of dioxopiperazine rings. (Witiak et al, J. Med. Chem., (1985), 28, 1228-1234.)
A major problem with bis(2,6-dioxopiperazine) drugs is their poor water solubility. For example, razoxane has a solubility of only 3 mg/ml in water at 25.degree. C. (Ren et al, Kuexue Tongbao, (1980), 25, pp. 189-190). Use of cosolvents, complexation, or crystalline modification to overcome low water solubility problems has not been successful. Interestingly, resolution provides enantiomers of razoxane, namely (+) ICRF-187 and (-) ICRF-186, which possess significantly greater water solubility than the racemic material. Additionally, bis(morpholinomethyl) derivatives such as bimolane (29), a derivative of ICRF-154, has increased water solubility. (Ren et al, Kuexue Tongbao, (1980), 25, 189-190). Insertion of morpholinomethyl functions into dioxopiperazines does not always afford water soluble materials. The bis(morpholinomethyl) analogue of ICRF-159 (razoxane)(30), also known as probimane, is more water soluble than bimolane (Witiak and Bhat, U.S. Pat. No. 4,871,736 issued Oct. 3, 1989). Morpholinomethyl derivatives of tricyclic analogues 22 and 23, however, are insoluble. (Witiak et al, J. Med. Chem., (1985), 28, 1228-1234). ##STR8##
Other available derivatives (31-33) result from substitution of imide nitrogens with maleimide, a sulfhydro reagent, maleic hydrazide, a plant growth inhibitor having weak antitumor activity, and 2-amino-1,3,4-thiadiazole, a weakly antineoplastic heterocycle. (Ren et al, Eur. J. Cancer Clin. Oncol., (1985), 21, 493-497). When compared for anti-tumor activity against leukemia L1210, S180, LL carcinoma, and Ridgway osteosarcoma, generally, bimolane and the maleic hydrazide analogue 32 have somewhat better properties. (Ren et al, Id.) ##STR9##
The most potent of many (30, 34-40) bimolane analogues known (Ren et al, 14th International Congress of Chemotherapy, Jun. 23-28, 1985, Kyoto, Japan; He et al, Zhongguo Xaoli Xuebao, (1988), 9, 369-373; Huang et al, Zhongguo Xaoli Xuebao, (1984), 5, 69-71) is probimane (30). Unlike razoxane, probimane may be readily administered parenterally. (Herman et al, Cancer Chemother. Pharmacol., (1987), 19, 277-281). ##STR10##
Bimolane is unstable, and the antitumor properties exhibited by this compound may reflect chemical hydrolysis or metabolism to ICRF-154 in biological systems. (Camerman et al, Science, (1984), 225, 1165-1166). However, bis(morpholinomethyl) derivatives of tricylic bis(imide)s 18-23 have activities which differ from the parent drugs. This suggests that the bis(morpholinomethyl) species may have intrinsic stereoselective antineoplastic and antimetastatic properties. Perhaps, solubility and transport differences for the various derivatized stereoisomers may be responsible for stereoselective differences in biological activities. (Herman et al, Adv. Pharmacol. Chemother., (1982), 19, 249-290).
Morpholinomethyl derivatives may be easily prepared from the parent bis(dioxopiperazines)s by reaction with morpholine and formaldehyde, and are of interest because of the important clinical results observed for bimolane. Comparative analysis of bis(morpholinomethyl) derivatives of tricycles 20 and 21 with their respective parent bis(imide)s for antitumor effects in mice using a postamputation schedule LL model has shown the bis(morpholinomethyl) derivative of cis-21 to be the most effective inhibitor of metastasis. (Witiak et al, J. Med. Chem., (1985), 28, 1111-1113). However, the bis(morpholinomethyl) derivative of trans-20 also exhibits considerable antimetastatic activity and is more effective than the parent trans bis(imide) 20. The cis dioxopiperazine 21 is a better inhibitor of metastasis than trans-20 in this assay. However, in the B16-F10 model, trans-20 has greater antimetastatic activity than cis-21. (Witiak et al, J. Med. Chem., (1984), 24, 1329-1332). Examination of regioisomeric bis(imides) 22 and 23 and their corresponding bis(morpholinomethyl) derivatives in the LL model reveals that only the morpholinomethyl derivative of cis-23 inhibits metastasis. (Witiak et al, J. Med. Chem., (1985), 28, 1228-1234). Since the parent bis(imide) cis-23 does not exhibit any appreciable antimetastatic effect in this assay, it seems unlikely that the effect of bis(morpholino)-23 is due to its hydrolysis to cis-23. Possibly, but not conclusively, morpholinomethyl derivatives of bis(dioxopiperazine)s possess intrinsic antitumor properties independent of their obvious instability in aqueous systems and their potential as prodrugs.
Many N-acyloxymethyl derivatives (41-51) of ICRF-154 are available. (Cai et al, 14th Intl. Cong. Chemother., Jun. 23, 1985, Kyoto, Japan). Antitumor activities depend upon the nature of the acyl moiety. Compound 50 (MST-16) is the most promising antitumor drug among these derivatives, and is more effective than ICRF-154 and razoxane against Pb388, L1210 and B16 tumors. (Cai et al, Id.). The structure of compounds 41-51 is set forth below: ##STR11##
Additionally, the synthesis of bis(morpholinomethyl) derivatives of bis(dioxopiperazine)s is disclosed in U.S. Pat. No. 5,130,426, and U.S. Pat. No. 4,950,755, all by Witiak et al, which applications and patents are all incorporated by reference herein.
The synthesis of stereoisomeric tricyclic bis(dioxopiperazines) is also disclosed in U.S. Ser. No. 596,364 filed on Apr. 3, 1984 by Nair et al and U.S. Pat. No. 4,871,736 by Nair et al, which are also incorporated by reference herein. The use of these compounds as antitumor and antimetastatic agents is also taught.
The synthesis of various diastereomeric mono- and di-hydroxylated diamino cyclohexane compounds, in particular, cyclohexane-1,2-di(O--)-4,5-di(N)diastereomers, and the use of such compounds as synthons in the preparation of antitumor platinum complexes is also known as disclosed by Witiak et al, in U.S. Pat. No. 5,206,400, which is also incorporated by reference in its entirety herein.
Recently, Witiak et al, in the "Synthesis and .sup.1 HNMR Conformational Analyses of Diastereomeric 4,4'-(4,5-Dihydroxy-1,2-cyclohexanediyl)bis-2,6-piperazinediones and a Synthetically Related Tricyclic Octahydro-2,2dimethyl-6-oxo-1,3-dioxolo 4,5-g! quinoxaline 5,8-diacetic Acid Ester" submitted to Witiak and Wei, J. Org. Chem., (1991), 56, 5408-5417, and Wei, Yung, Ph.D. Thesis, Ohio State University, 1990, disclosed the preparation of novel diasteriomeric 4,4'-(4,5-dihydroxy-1,2-cyclohexanediyl)bis (2,6-dioxopiperazine)s compounds (88-93) and the synthetically related tricyclic 1,3-dioxolo4,5-g!quinoxaline ring system compound (86) from their respective (4,5-dihydroxy-1,2cyclohexanediyl)bis(carbamate)s compounds (52-57) via isopropylidene-protected intermediates (58-63). These compounds (88-93) have two hydroxyl groups introduced on the 4 and 5 positions of the cyclohexane-1,2-diyl bis(dioxopiperazine) system were proposed to increase intermolecular hydrogen bonding and to enhance aqueous solubility. The compounds are set forth on the following page: ##STR12## The preparation of these compounds is disclosed in Witiak et al, Id. and Wei, Id., which are incorporated by reference in their entirety.
Essentially, known diastereomeric dihydroxy cyclohexanediyl bis(carbamate)s (52-57), were protected as their isopropylidene ketals (58-63), and served as precursors to the intermediate isomeric diamines, five of which (64-68) were convertible to targets (88-93) via tetra (esters) (70-74) and bis(dioxopiperazines) (Witiak et al, Id. and Wei, Id.).sup.1. The reaction scheme is set forth on the following page: FNT .sup.1 The structures for compounds 52-80 may be found in Chart I which is on the following page. ##STR13##
CHART I __________________________________________________________________________ ##STR14## ##STR15## 52, R.sub.1 = R.sub.2 = NHCBZ, R.sub.3 = R.sub.4 = H 53, R.sub.1 = R.sub.2 = NHCBZ, R.sub.3 =!R.sub.4 54, ##STR16## NHCBZ, R.sub.3 = R.sub.4 = H 58, R.sub.1 = R.sub.2 = NHCBZ, 59, R.sub.1 = R.sub.2 = NHCBZ, 60, R.sub.1 = R.sub.2 = NHCBZ, R.sub.3 = R.sub.4 = Me.sub.2 C R.sub.3 = R.sub.4 = Me.sub.2 C R.sub.3 = R.sub.4 = Me.sub.2 C 64, R.sub.1 = R.sub.2 = NH.sub.2, 65, R.sub.1 = R.sub.2 = NH.sub.2, 66, R.sub.1 = R.sub.2 = NH.sub.2, R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C 70, R.sub.1 = R.sub.2 = N(CH.sub.2 CO.sub.2 Et).sub.2 71, R.sub.1 = R.sub.2 = N(CH.sub.2 CO.sub.2 Et).sub.2 72, R.sub.1 = R.sub.2 = N(CH.sub.2 CO.sub.2 Et).sub.2 R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C 76, ##STR17## 77, ##STR18## 78, ##STR19## R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C ##STR20## ##STR21## ##STR22## 55, R.sub.1 = R.sub.2 = NHCBZ, 56, R.sub.1 = R.sub.2 = NHCBZ, 57, R.sub.1 = R.sub.2 = NHCBZ, R.sub.3 = R.sub.4 = H R.sub.3 = R.sub.4 = H R.sub.3 = R.sub.4 = H 61, R.sub.1 = R.sub.2 = NHCBZ, 62, R.sub.1 = R.sub.2 = NHCBZ, 63, R.sub.1 = R.sub.2 = NHCBZ, R.sub.3 = R.sub.4 = Me.sub.2 C R.sub.3 = R.sub.4 = Me.sub.2 C R.sub.3 = R.sub.4 = Me.sub.2 C 67, R.sub.1 = R.sub.2 = NH.sub.2, 68, R.sub.1 = R.sub.2 = NH.sub.2, 69, R.sub.1 = R.sub.2 = NH.sub.2, R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C 73, R.sub.1 = R.sub.2 = N(CH.sub.2 CO.sub.2 Et).sub.2 74, R.sub.1 = R.sub.2 = N(CH.sub.2 CO.sub.2 Et).sub.2 75, R.sub.1 = R.sub.2 = N(CH.sub.2 CO.sub.2 Et).sub.2 R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C 79, ##STR23## 80, ##STR24## R.sub.3, R.sub.4 = Me.sub.2 C R.sub.3, R.sub.4 = Me.sub.2 C __________________________________________________________________________
Conversion of diol (52) to the corresponding acetonide (58) using acetone-perchloric acid or trimethylsilyl enol ether was unsuccessful. However, all six protected diastereomers (58-63) were easily prepared employing 2-methoxypropene (2-MP) in TsOH-acetone rather than DMF using the procedures of Fanton et al, J. Org. Chem., (1981), 46, 4007, and Sommer et al, J. Org. Chem., (1971), 36, 82. The individual diastereomer derivatives were obtained in 81-95% yields with relatively minor modifications in the experimental detail such as (a) molar ratios of 2-MP to starting diol, (b) reaction temperatures, and (c) reaction times. The exact details of these modifications may be found in Witiak et al., (Id.).
The diastereomeric diamines (64-69) were obtained in virtually quantitative yield upon catalytic (10% Pd/c) hydrogenation (20 psi; MeOH) of the respective bis(carbamate)s (58-63). However, undesired tricyclic acetone-derived imidazolidines were generated from cis bis(carbamate) diastereomers (58, 59 and 63) during solvent removal under reduced pressure and at room temperature. Imidazolidine formation, however, was precluded when the reagent grade MeOH is replaced by HPLC-grade MeOH which contains&lt;0.001% acetone. All diamines were used without further purification, and five of six compounds, (64-68), underwent tetra(N-alkylation) at room temperature (24 hours) and in 53-77% yield with ethylbromoacetate in DMF containing 1,2,2,6,6-pentamethylpiperazine (PMP). The purification of the tetraesters (70-74) was effected by silica gel chromatography using hexane ethyl acetate (2:1) as an eluant. However, diastereomer (72) was purified using a large diameter-short length column with the addition of 1 drop of triethylamine to each 3 mL of eluant, apparently because of considerable tailing of the diastereomer.
Additionally, since tetra(ester) (71) was obtained from diamine (65) in only 53% yield, Witiak et al developed an alternative synthesis (Scheme II) set forth below: ##STR25## comprising catalytic osmylation of tetra(ester)(82) prepared from the diamine (81). The resulting diol (83), produced in 70% yield, was apparently uncontaminated from the isomer resulting from reaction on the opposite side of the double bond. However, further purification was effected by silica gel chromatography using CHCl.sub.3 /acetone (3:1) as an eluant. Conversion to acetonitrile (71) was accomplished in approximately 100% yield using 2-MP in TsOH-acetone, confirming the stereochemical assignment for intermediate (83).
The desired tetra(ester) (75) could not be obtained under the experimental conditions used for producing the other five tetra(ester) diastereomers (70-74). Instead, the diamine (69) reacted with three moles of ethyl .alpha.-bromoacetate and subsequently underwent intramolecular cyclization to produce either tricyclic trans-anti-cis lactam (86) or the regioisomeric trans-anti-cis lactam (87) likely via intermediates (84) or (85), respectively. This reaction scheme is depicted in Scheme III on the following page: ##STR26##
Among the five bis(2,6-dioxopiperazine) diastereomers synthesized, only cis-anti-cis diastereomer (89) exhibits enhanced water solubility (29.3 mg/ml at 25.degree. C.). Wei et al speculate therein that differences in crystal packing, in addition to the relative competition between intra- and intermolecular hydrogen bonding may affect water solubility. It is further hypothesized that the unique 1,4 trans relationship of dioxopiperazine and hydroxyl groups found in this molecule may provide for enhanced water solubility and also reduced melting point because of the loose crystal packing of this conformationally flexible species.
From the above discussion it is clear that many dioxopiperazines are known in the art, and have been utilized as therapeutic agents. However, to date, the exact biological mechanism(s) by which bis(dioxopiperazine)s mediate therapeutic efficacy remains uncertain.
Various groups have studied the biological effects of bis(dioxopiperazine)s in an effort to determine the mode of action for this large class of compounds. These studies have predominantly focused on razoxane, a bis(dioxopiperazine) which is currently used in cancer treatment. Various theories have been advanced as to the biological mechanism by which bis(dioxopiperazine)s mediate therapeutic efficacy. For example, Sharpe et al, Nature, (1970), 226, 524, and Hellman et al., J. Nat'l Cancer Inst. (1970), 44, 539-543 purport that razoxane mediates a cytostatic effect by inhibiting the transition from G.sub.2 to M in the cell cycle. Related to this, Atherton et al, British J. of Derm., (1980), 102, 307 also disclose that razoxane apparently inhibits cell cycle processing during the late pre-mitotic (G.sub.2) or early mitotic (M) phase of the cell cycle making this compound potentially applicable in the treatment of psoriasis.
Another possible biological mechanism explaining the therapeutic activity of bis(dioxopiperazine)s involves their "normalization" of tumor cell vasculature. For example, Serve et al., British Medical Journal, (1972), 1, 597-601; James et al, Cancer Research, (1974), 34, 839-842; and Salsbury et al, Cancer Research, (1974), 34, 843-849 disclose that razoxane induces striking changes in the morphology and physiology of tumor vasculature which results in their appearance and behavior resembling that of normal blood cells. It is suggested that these changes may explain the antimetastatic effect of razoxane. This theory finds support, e.g., in Olweny et al, Cancer Treatment Reports, (1976), 60(1); 111-113 who disclose the treatment of Kaposi's sarcoma, a highly vascular sarcoma, with razoxane and teach the antimitotic effects and vascular normalizing activity of razoxane.
Another possible explanation for the therapeutic effects of bis(dioxopiperazine)s involves their chelating activity. For example, Herman et al, Cancer Chemother. Pharmacol., (1987), 19, 277-281, summarize that a bis(morpholinomethyl) derivative of razoxane likely inhibits chronic doxorubicin cardiotoxicity because of its ability to chelate iron thereby inhibiting the formation of iron-doxorubicin complexes which result in highly reactive and toxic oxygen-containing free radicals.
Yet another theory explaining the therapeutic activity of bis(dioxopiperazine)s involves their putative inhibition of mammalian DNA topoisomerase II. DNA topoisomerases are the enzymes involved in the conversion of DNA topology which are essential for many genetic processes. Recently, many antitumor agents have been shown to be topoisomerase-targeting drugs. Tanabe et al, Cancer Research, (1991), 51, pp. 4903-4908 and Ishida et al., Cancer Research, (1991), 51, 4909-4916 disclose that bis(2,6-dioxopiperazine)s inhibit topoisomerase II by preventing etoposide-induced cleavable complex formation and that this inhibition may relate to the abnormal appearance of cells in the G.sub.2 and early M phase of the cell cycle.
Still another theory explaining the antitumor activity of razoxane involves its possible effects on basement membrane collagen degradation. For example, Karakiulakis et al, Meth Find. Exp. Clin. Pharmacol., (1989), 11(4), 255-261, hypothesize that razoxane inhibits degradation of intact basement membrane or of type IV collagen. However, inhibition was not proven since an ammonium sulfate enzyme extract obtained from Walker 256 carcinosarcoma was used rather than a pure enzyme. Another group, Boggust et al, British J. Cancer, (1978), 38, 329-334 purport that collagen-peptidase activity in HeLa cell extracts and human tumors is inactivated by razoxane and that this inhibition may be involved in the prevention of metastasis. However, this study was also inconclusive since crude HeLa cell extracts were utilized rather than purified enzymes.
Also, Duncan et al, Biochemical Pharmacol., (1983), 32 (24), 3853-3858 teach that razoxane inhibits the production of collagenases and specific tissues inhibitor of metalloproteinase (TIMP) by stimulated articular chondrocyes, and that this may be involved in the therapeutic efficacy of razoxane for treatment of psoriatic arthritis.
Yet another theory explaining the efficacy of bis(dioxopiperazine)s is that they exhibit antitumor activity by mediating a combination of several independent functions, including, e.g., the inhibition of enzymes; cytostatic or cytotoxic action; and immunosuppressive effects. For example, Boggust, Excerpta Medica, (1978), 4, 106-112 disclose that the antitumor activity of razoxane may be attributable to a combination of effects which include: (1) effect on growth and metabolism of cancer cells in primary tumors, (2) effect upon enzymes and other factors which regulate tumor cell retention and release, (3) effect on viability of malignant cells within vessels of the circulatory system, (4) effect on enzymes which degrade structural components of the capillary wall and endothelial intercellular cement (basement membrane), (5) release of factors from tumor which regulate capillary growth and formation and (6) effect on growth of capillary endothelial cells stimulated by tumor growth promoters.
Thus, while there exist many theories as to how bis(dioxopiperazine)s mediate therapeutic effects, a conclusive biological mechanism(s) which explains, or is at least predicative as to the therapeutic efficacy of a particular bis(dioxopiperazine) compound, is currently unavailable.